Adult Raphe-Specific Deletion of Lmx1b Leads to CentralSerotonin DeficiencyNing-Ning Song1*, Jian-Bo Xiu1, Ying Huang1, Jia-Yin Chen1, Lei Zhang1, Lise Gutknecht2, Klaus Peter
Lesch2, He Li3, Yu-Qiang Ding1*
1 Department of Anatomy and Neurobiology, Tongji University School of Medicine, Shanghai, China, 2 Molecular and Clinical Psychobiology, Department of Psychiatry
and Psychotherapy, University of Wurzburg, Wurzburg, Germany, 3 Key Laboratory of Nervous System Diseases, Ministry of Education of China, Division of Histology and
Embryology, Department of Anatomy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Abstract
The transcription factor Lmx1b is essential for the differentiation and survival of central serotonergic (5-HTergic) neuronsduring embryonic development. However, the role of Lmx1b in adult 5-HTergic neurons is unknown. We used an inducibleCre-LoxP system to selectively inactivate Lmx1b expression in the raphe nuclei of adult mice. Pet1-CreERT2 mice weregenerated and crossed with Lmx1bflox/flox mice to obtain Pet1-CreERT2; Lmx1bflox/flox mice (which termed as Lmx1b iCKO).After administration of tamoxifen, the level of 5-HT in the brain of Lmx1b iCKO mice was reduced to 60% of that in controlmice, and the expression of tryptophan hydroxylase 2 (Tph2), serotonin transporter (Sert) and vesicular monoaminetransporter 2 (Vmat2) was greatly down-regulated. On the other hand, the expression of dopamine and norepinephrine aswell as aromatic L-amino acid decarboxylase (Aadc) and Pet1 was unchanged. Our results reveal that Lmx1b is required forthe biosynthesis of 5-HT in adult mouse brain, and it may be involved in maintaining normal functions of central 5-HTergicneurons by regulating the expression of Tph2, Sert and Vmat2.
Citation: Song N-N, Xiu J-B, Huang Y, Chen J-Y, Zhang L, et al. (2011) Adult Raphe-Specific Deletion of Lmx1b Leads to Central Serotonin Deficiency. PLoSONE 6(1): e15998. doi:10.1371/journal.pone.0015998
Editor: Etienne Joly, Universite de Toulouse, France
Received September 9, 2010; Accepted December 2, 2010; Published January 5, 2011
Copyright: � 2011 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Natural Science Foundation of China (30430260), the Ministry of Science and Technology of China(2006CB943903, 2007CB512303, 2009ZX09501-030) and the Deutsche Forschungsgemeinschaft (KFO 125, SFB 581, SFB TRR 58). The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (N-NS); [email protected] (Y-QD)
Introduction
The neurotransmitter serotonin (5-HT) exerts a wide spectrum of
actions in a variety of behaviors, such as pain sensation, locomotion,
circadian rhythm, food intake and emotional behaviors [1,2].
Extensive efforts have been made to characterize the molecular
pathways that control the specification, differentiation and survival of
5-HTergic neurons during brain development [3], because this line of
research is very helpful for understanding the genetic basis of central 5-
HT deficiency which leads to many mental disorders [4,5]. Sonic
hedgehog secreted from the floor plate triggers the expression of
Mash1 and GATA2 in progenitor cells in the ventricular zone of
hindbrain [6], and both genes are essential for the development of 5-
HTergic neurons [7,8]. 5-HTergic neurons are classified into two
groups based on their anatomical location: a rostral group located in
the pons and a caudal group located in the medulla oblongata.
Although Nkx2.2 is expressed in the progenitors of all 5-HTergic
neurons in hindbrain, evidence from null mutant mice show that it is
only required for the generation of 5-HTergic neurons in the dorsal
raphe nucleus, one cluster neurons in pons group [9], and GATA3 is
thought to be required for the differentiation of the medulla oblongata
group [10]. Both Lmx1b and Pet1 are expressed in postmitotic 5-
HTergic neurons and essential for the differentiation and survival of 5-
HTergic neurons during embryonic development [4,11,12].
Our previous study has shown that Lmx1b is persistently
expressed in central 5-HTergic neurons during postnatal devel-
opment and throughout adulthood suggesting that Lmx1b may be
involved in regulating normal expression of 5-HT in adult brain.
To test this hypothesis, we used a tamoxifen-inducible Cre-LoxP
system [13] to selectively inactivate Lmx1b expression in central 5-
HTergic neurons of adult mice. Our data showed that 5-HT level
in Lmx1b iCKO mice was reduced to 60% of control mice
probably due to down-regulation of Tph2. In addition, Sert and
Vmat2 that are implicated in maintaining normal functions of 5-
HTergic neurons were greatly reduced in Lmx1b iCKO mice.
Thus, Lmx1b, an essential gene for the development of central 5-
HTergic neurons, is also required for the normal biosynthesis of 5-
HT in the adult brain and possibly for regulating normal functions
of central 5-HTergic neurons.
Materials and Methods
Genetic crossings, genotyping and animal maintenanceLmx1bflox/flox mice [14] and Rosa26-LacZ reporter (Rosa26R)
mice [15] were generated and genotyped as previously described.
In Lmx1bflox/flox mice, exons 4-6 of Lmx1b were flanked by two
LoxP sites and can be deleted in the presence of Cre in vivo [14].
To specifically inactivate Lmx1b expression in 5-HTergic neurons
in the adult mouse brain, Lmx1bflox/flox mice were crossed with
Pet1-CreERT2 mice (see below) and their offspring Pet1-CreERT2;
Lmx1bflox/+ mice were then crossed with one another to obtain
Pet1-CreERT2; Lmx1bflox/flox (Lmx1b iCKO) mice. Animal care
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practices and all experiments were reviewed and approved by the
Animal Committee of Tongji University School of Medicine,
Shanghai, China (TJmed-010-10).
Generation of Pet1-CreERT2 micePet1-CreERT2 BAC construct was obtained by inserting
CreERT2 coding sequence downstream of the Pet1 start codon
within the RP23-165D11 BAC (BACPAC Resources Center at
Children’s Hospital Oakland Research Institute) via homologous
recombination in EL250 bacteria [16]. Sepharose-4B (Sigma)-
purified BAC DNA was then introduced into FVB/N fertilized
mouse eggs by pronuclear injection using standard methods.
Transgenic mice were genotyped by PCR with primers
against Cre (forward: TCG ATG CAA CGA GTG ATG AG;
reverse: TCC ATG AGT GAA CGA ACC TG) resulting in a
,400 base-pair product. All progeny carrying this transgene
were found to be viable and fertile without any obvious
abnormalities.
To determine the spatial pattern of Cre activity, Pet1-CreERT2
mice were crossed with Rosa26R mice [15] and Cre activity was
examined by administering tamoxifen to Pet1-CreERT2; Rosa26R
progeny. Tamoxifen (20 mg/ml; Sigma) diluted in corn oil (Sigma)
was administered by oral gavage in once-daily doses of 8 mg/40 g
of body weight on the following schedule: days 1, 8, 9, 11 and 12.
Mice were sacrificed 2-3 weeks after the last dose and brains were
removed and fixed in 4% paraformaldehyde (Sigma) in 0.01 M
phosphate buffered saline (PBS; pH 7.4) for 3 hours. After
cryoprotection with 30% sucrose in PBS, 40 mm-thick sections
were cut on a cryostat (CM1900, Leica) and immediately
subjected to X-gal staining as described previously [5].
In situ hybridization and immunohistochemistryIn situ hybridization probes against Tph2, Sert, Aadc and Dopamine
b-hydroxylase (Dbh), were constructed according to the description
on the website of Allen Brain Atlas (http://www.brain-map.org).
The Lmx1b [17] and Pet1 in situ probes encompassed the complete
Figure 1. Cre-reombinase activity in Pet1-CreERT2 mice. (A) Diagram of the Pet1-CreERT2 construct. The CreERT2 coding sequence was insertedin frame into a BAC construct downstream of the Pet1 start codon by homologous recombination. (B) The procedure for tamoxifen administration.Tamoxifen was administrated on D1, D8, D9, D11 and D12 and mice were sacrificed for analysis 2–3 weeks after the last dose.(C–G). Distribution of X-gal-positive cells in the raphe nuclei of Pet1-CreERT2; Rosa26R mice. (H–J). No X-gal positive cells were found in other brain regions. Ctx, cerebralcortex; DR, dorsal raphe nucleus; Hip, hippocampus; Hypo, hypothalamus; LPGi, lateral paragigantocellular nucleus; MM, medial mammillary nucleus;MnR, median raphe nucleus; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; SN, substantia nigra. Scale bars,100 mm (B–F) and 200 mm (G–I).doi:10.1371/journal.pone.0015998.g001
Adult Deletion of Lmx1b Leads to Central 5-HT Loss
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Lmx1b and Pet1 coding sequence, respectively. We also generated
an in situ probe against exons 4–6 of Lmx1b only. All probes were
cloned into pGEM-T vector (Promega). Eight mice (4 wild-type
and 4 Lmx1b iCKO) were used for in situ hybridization. For in situ
hybridization, brains were fixed in 4% PFA in PBS for 24 hours,
cryoprotected with 30% sucrose in PBS, and 30 mm-thick
transverse sections were cut on a cryostat and mounted onto glass
slides (Fisher Scientific). RNA probes labeled by digoxygenin-UTP
(Roche) were generated by in vitro transcription and hybridization
signals were visualized upon nitro blue tetrazolium chloride
(Fermentas) and 5-bromo-4-chloro-3-indolyl phosphate (Fermen-
tas) staining.
Twelve mice (6 wild-type and 6 Lmx1b iCKO) were used in
immunochemistry. Thirty mm-thick brain sections were incubated
with primary antibody at 4uC overnight. After washing in PBS,
sections were incubated with appropriate secondary antibody for
3 hours at room temperature, washed in PBS, and incubated with
Cy3-conjugated streptavidin (1:1000; Jackson ImmunoResearch)
for 1 hour. The following primary antibodies were used: goat anti-
b-galactosidase (b-gal; 1:1000; AbD Serotec), rabbit anti-Lmx1b
(1:2000) [17], rabbit anti-Tph2 (1:4000) [18,19], rabbit anti-Vmat2
(1:1000; Chemicon), mouse anti-Tyrosine hydroxylase (TH; 1:4000;
Sigma). For Tph2 and b-gal double staining, sections were
incubated with a mix of the anti-Tph2 and anti-b-gal antibodies
overnight, then for 3 hours with a mix of Cy3-labeled donkey anti-
rabbit (1:400; Jackson ImmunoResearch) and biotinylated horse
anti-goat IgG (1:400; Vector Laboratories), and finally with Cy2-
conjugated streptavidin (1:1000; Jackson ImmunoResearch) for
1 hour. There were no immunostaining signals when primary
antibodies were omitted or replaced with normal IgG. Stained
sections were observed and scanned under a fluorescence or
confocal microscope.
Figure 2. Pet1-CreERT2 activity is specific to central 5-HTergic neurons. Double immunolabeling of Tph2 and b-gal in the dorsal raphenucleus (DR; A-B’’) and raphe magnus nucleus (RMg; C-C’’) was performed in Pet1-CreERT2; Rosa26R mice. About 85% of Tph2-positive neurons are co-stained with b-gal antibody (arrows), and a few Tph2-labeled neurons are not b-gal positive (arrowhead). Note that all b-gal-expressing neurons arelabeled with Tph2 antibody. B-B’’ show higher magnifications views of A-A’’, respectively. Scale bars, 100 mm.doi:10.1371/journal.pone.0015998.g002
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Cell countWe counted positive cells in every six sections. Positive cells in the
dorsal raphe nucleus (around the level of 24.72 mm to Bregma)
[20] and raphe magnus nucleus (around the level of 24.84 mm to
Bregma) were counted for statistical comparison between wild-type
and Lmx1b iCKO mice (n = 4 for each). Statistical significance was
determined by the Mann-Whitney test. All data were expressed as
mean 6 SEM, and error bars represent SEM. P values less than
0.05 were considered statistically significant.
High performance liquid chromatography (HPLC)Adult (3 months) tamoxifen-induced wild-type and Lmx1b
iCKO mice were used for HPLC (n = 6 for each). Two-three
weeks after completion of tamoxifen treatment, whole brains were
dissected out immediately after anesthesia with sodium pentobar-
bital (0.07 mg/g body weight), and HPLC samples were made
according to methods described previously [5]. 5-HT and its
metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopamine and
its metabolite dihydroxyphe-nyacetic acid and homovanillic acid,
and norepinephrine were measured using HPLC electrochemical
detection as described previously [5]. Statistical significance was
determined by the Student’s t-test. All data were expressed as
mean 6 SEM, and error bars represent SEM. P values less than
0.05 were considered statistically significant.
Results
Generation and characterization of Pet1-CreERT2
transgenic miceTo delete Lmx1b in the adult mouse brain, we crossed mice
carrying two floxed Lmx1b alleles with mice harboring a tamoxifen-
inducible form of Cre recombinase (CreERT2) [13] under the
control of the Pet1 promoter, to generate Lmx1b iCKO mice
(Figure 1A). Genotypes were confirmed by PCR. Pet1 is expressed
specifically in central 5-HTergic neurons [4], and the distribution of
Cre-recombination activity was determined by crossing to Rosa26R
mice [15]. Pet1-CreERT2; Rosa26R mice were administered a
Figure 3. Lmx1b is required for the normal expression of 5-HTin the adult brain. (A, B) Intensities of immunofluorescence in most 5-HT-positive neurons in the dorsal raphe nucleus (DR) are reduced inLmx1b iCKO mice (B) relative to that in wild-type controls (A). (C, D)Similar changes in intensities of 5-HT immnofluorescence are alsopresent in the raphe obscurus nucleus (Rob), raphe pallidus nucleus(RPa) and lateral paragigantocellular nucleus (LPGi) of Lmx1b iCKO micerelative to those of wild-type mice (C, D). (E) HPLC analysis showing thatlevel of 5-HT and its metabolite 5-HIAA in the brains of Lmx1b iCKOmice are reduced to about 60% and 30% of that in controls, respectively(* P,0.001). Scale bar, 100 mm.doi:10.1371/journal.pone.0015998.g003
Figure 4. Tph2 expression is reduced in adult Lmx1b iCKO mice.(A–D) The number of DR neurons strongly labeled with Tph2 (arrows inC, D) is decreased in Lmx1b iCKO mice (B, D) compared with wild-typecontrols (A, C). C and D are high magnifications of the boxed areas in Aand B, respectively. Note that most Tph2-positive neurons in Lmx1biCKO mice are only weakly labeled (arrowheads in D). (E, F) Similarresults are observed in the raphe obscurus nucleus (Rob), raphe pallidusnucleus (RPa) and lateral paragigantocellular nucleus (LPGi) of themedulla oblongata. Scale bars, 100 mm.doi:10.1371/journal.pone.0015998.g004
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regimen of tamoxifen (see Materials and methods) beginning at P90
and analyzed by X-gal staining 2–3 weeks after completing the
induction regimen. The procedure of tamoxifen administration is
shown in Figure 1B. The first day of tamoxifen administration was
termed as D1, and tamoxifen was administrated in once-daily doses
of 8 mg/40 g body weight on D1, D8, D9, D11 and D12. X-gal-
positive cells were found in the raphe nuclei and ventrolateral
reticular formation of the medulla oblongata (Figure 1C–G),
showing that Pet1-driven Cre-recombinase activity can be induced
in adulthood. Note that X-gal labeling was not observed in other
brain regions outside the raphe nuclei (Figure 1H–J) and no Cre
activity was present in negative control-treated and untreated Pet1-
CreERT2; Rosa26R mice (data not shown).
To determine whether Cre-recombinase activity in Pet1-
CreERT2; Rosa26R mice was exclusive to 5-HTergic neurons, we
performed Tph2/b-gal double immunostaining. Tph2 is the key
enzyme responsible for 5-HT synthesis in the brain [21]. All b-gal-
positive neurons expressed Tph2, and approximately 85% of
Figure 5. Decreased expression of 5-HT-specific genes in adult Lmx1b iCKO mice. (A–L) Tph2 (A–D) and Sert [34] in situ hybridization, andVmat2 (I–L) immunostaining shows down-regulation of these three genes in rostral and caudal raphe nuclei of Lmx1b iCKO mice (B, D, F, H, J, L)compared with that in wild-type controls (A, C, E, G, I, K). (M–T) Aadc (M–P) and Pet1 (Q–T) expression is unchanged in Lmx1b iCKO raphe nuclei (N, P,R, T) relative to wild-type controls (M, N, Q, S). DR, dorsal raphe nucleus; RMg, raphe magnus nucleus. Scale bar, 100 mm.doi:10.1371/journal.pone.0015998.g005
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Tph2-labeled neurons were b-gal-positive (Figure 2). Thus, Cre
activity in Pet1-CreERT2 mice is specific to 5-HTergic neurons, and
is capable of inducing recombination in the majority of 5-HTergic
neurons in the adult brain.
Deleting Lmx1b in the adult brain leads to 5-HTinsufficiency
Previous studies have demonstrated that Lmx1b is required for
the differentiation and survival of central 5-HTergic neurons
during embryonic development [11,12]. We set out to examine
whether Lmx1b also plays a role in the adult central 5-HTergic
neurons. Lmx1b iCKO and wild-type control mice were admin-
istrated with tamoxifen beginning at P90 and examinations were
performed 2–3 weeks after completion. We first used Lmx1b
antibody [17] to examine whether Lmx1b is deleted in Lmx1b
iCKO mice. As shown in Figure S1, similar Lmx1b immuno-
staining was found in the dorsal raphe nucleus of wild-type mice
and Lmx1b iCKO mice showing that the antibody recognizes the
truncated Lmx1b protein without exons 4–6 of Lmx1b; in this case
the Lmx1b antibody can be used to trace Lmx1b mutant cells.
Because the full length of in situ hybridization probe for Lmx1b [17]
was unable to distinguish truncated mRNA from normal Lmx1b
mRNA either (data not shown), we generated an in situ probe
against exons 4–6 of Lmx1b only. However, the sensitivity of this
probe was too low to detect Lmx1b mRNA in adult wild-type mice,
although it worked in showing Lmx1b mRNA in embryos (data not
shown). The floxed Lmx1b alleles were deleted after crossing
Lmx1bflox/flox mice with Pet1-Cre or Wnt1-Cre mice [5,14] and Cre
activity in Pet1-CreERT2 mice was functional as shown by X-gal
staining in 5-HTergic neurons in Pet1-CreERT2; Rosa26R mice
(Figures 1, 2), we thus speculate that the exons 4–6 of Lmx1b
should be deleted in the majority of 5-HTergic neurons in the
adult Lmx1b iCKO mice (also see phenotypes described below).
We next examined 5-HT expression in Lmx1b iCKO mice, and
found that intensities of 5-HT immunofluorescence in individual
neurons were slightly reduced in the raphe nuclei of Lmx1b iCKO
mice compared with wild-type mice (Figure 3A–D). To further
investigate whether the content of 5-HT in the brain is altered or
not, we used HPLC to measure the levels of 5-HT and its
metabolite 5-HIAA in the brain, and found that they were
decreased in Lmx1b iCKO mice to about 60% and 30% of control
levels, respectively (Figure 3E). We speculate that the discrepancies
between no apparent reduction of 5-HT immunofluorescence and
40% reduction of 5-HT revealed by HPLC in Lmx1b iCKO are
probably due to the low sensitivity of 5-HT antibody, which is
unable to detect this reduction. Taken together, we conclude that
Lmx1b is required for normal expression of 5-HT in adult brain.
Deleting Lmx1b in the adult brain results indown-regulation of 5-HTergic neuron-associated genes
To explore the mechanisms underlying decreased 5-HT level in
Lmx1b iCKO mice, we examined the expression of Tph2, which is
a specific enzyme for synthesis of 5-HT in the brain [21]. The
number of neurons with intense Tph2 immunofluorescence in the
raphe nuclei of Lmx1b iCKO mice was dramatically reduced
compared with control mice (Figure 4). Correspondingly, many
weakly-labeled neurons were seen in the Lmx1b iCKO raphe
nuclei (arrowheads in Figure 4D), whereas they were not observed
in wild-type mice. These observations were further confirmed by in
situ hybridization for Tph2 (Figure 5A–D). The number of cells
with intense in situ signals was significantly decreased in Lmx1b
iCKO relative to wild-type mice (Figure 6). Since approximately
15% of 5-HTergic neurons in Pet1-CreERT2 mice did not exhibit
Cre activity (Figure 2), the strong Tph2 labeling retained in Lmx1b
iCKO mice may correspond to 5-HTergic neurons in which
Lmx1b was not deleted. Nevertheless, these results indicate that
deleting Lmx1b in the adult brain impairs Tph2 expression, leading
to a deficiency of central 5-HT.
To further investigate the function of Lmx1b, we examined the
expression of several genes essential for maintaining the normal
function of 5-HTergic neurons. Sert is required for the re-uptake
of 5-HT in axonal terminals [22], and its expression was greatly
reduced in the raphe nuclei of Lmx1b iCKO mice (Figure 5E–H).
Cell counts showed a significant difference in the number of Sert-
expressing cells between wild-type and Lmx1b iCKO mice
(Figure 6). Vmat2, which is required for packaging 5-HT into
synaptic vesicles [23], was also down-regulated in Lmx1b iCKO
Figure 6. Decrease in numbers of Tph2- and Sert-labeled neurons in the dorsal raphe nucleus (DR) and raphe magnus nucleus(RMg). The number of neurons with intense in situ signals of Tph2 and Sert are significantly decreased in the DR (A) and RMg (B) of Lmx1b iCKO mice(n = 4; * P,0.05), while the number of Aadc positive cell is comparable in Lmx1b iCKO mice and wild-type mice (n = 4, P.0.5).doi:10.1371/journal.pone.0015998.g006
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mice (Figure 5I–L). These results indicate that expression of the
genes associated with the maintaining functions of 5-HTergic
neurons is impaired. To test whether deleting Lmx1b decreases the
number of 5-HTergic neurons or not, which in turn results in the
phenotypes mentioned above, we examined the expression of Aadc
and Pet1. We found that the number of Aadc-expressing neurons
was unchanged in Lmx1b iCKO mice compared with controls
(Figures 5M–P, 6), consistent with the finding that similar Lmx1b
immunostaining was present in both Lmx1b iCKO and wild-type
mice (Figure S1). The dorsal raphe nucleus contains the most
abundant 5-HTergic neurons among the raphe nuclei. In Nissl-
stained sections, the Nissl-stained 5-HTergic neurons are larger
and more intensely stained relative to non-5-HTergic neurons.
Nissl-stained sections from wild-type and Lmx1b iCKO mice
showed no obvious difference in cell density and distribution
(Figure S2). Furthermore, Pet1 expression in 5-HTergic neurons
requires Lmx1b during embryonic development [12], but its
expression in the raphe nuclei of Lmx1b iCKO mice showed no
difference from wild-type controls (Figure 5Q–T). These results
suggest that the overall number of 5-HTergic neurons is not
affected by deleting Lmx1b in adulthood, and that Pet1 expression
in the adult brain is independent of Lmx1b.
Expression of dopamine and norepinephrine isunchanged in Lmx1b iCKO mice
revious studies have shown that central 5-HT deficiency may
affect the expression of other monoamines in the brain [24]. To
explore this possibility, we examined the expression of TH, the
essential enzyme for the synthesis of both dopamine and norepine-
phrine, and Dbh, an enzyme that converts dopamine into
norepinephrine [25] in Lmx1b iCKO mice. TH immunostaining
in the substantia nigra and ventral tegmental area (dopaminergic
neurons), and in the locus coeruleus (norepinephrinergic neurons)
in Lmx1b iCKO mice was similar to that in control mice
(Figure 7A, B, D, E). Dbh in situ hybridization in the locus
coeruleus of Lmx1b iCKO mice was also similar to that of wild-
type controls (Figure 7C, F). In addition, levels of norepinephrine,
dopamine and its metabolites (dihydroxyphenylacetic acid and
Figure 7. Dopamine (DA) and norepinephrine (NE) expression is unchanged in adult Lmx1b iCKO mice. (A, B, D, E) TH immunostaining inthe substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) of the midbrain (A, D), and in the locus coeruleus (LC) of the pons (B, E) inLmx1b iCKO mice (D, E) is similar to that of wild-type mice (A, B). (C, F) In situ hybridization of Dbh in Lmx1b iCKO mice (F) is similar to that of wild-typecontrols (C). (G) Levels of DA, DA metabolites (dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA] and NE in the brains of Lmx1b iCKOmice are not different from those of control mice. Scale bar, 100 mm.doi:10.1371/journal.pone.0015998.g007
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homovanillic acid) in Lmx1b iCKO mice were not differ-
ent from those of controls, as determined by HPLC analysis
(Figure 7D). Thus, expression of dopamine and norepinephrine in
Lmx1b iCKO mice appeared normal.
Discussion
In the present study, we took advantage of an inducible Cre-
LoxP system to inactivate Lmx1b expression in adult 5-HTergic
neurons. We found that the level of central 5-HT in Lmx1b iCKO
mice is reduced to 60% of controls, and that the expression of 5-
HT neuron-associated genes such as Tph2, Sert and Vmat2 are
down-regulated in Lmx1b iCKO mice.
We generated Pet1-CreERT2 mice, and X-gal staining data from
Pet1-CreERT2; Rosa26R mice treated with tamoxifen in adulthood
showed that Cre was functional and restricted to central 5-
HTergic neurons. Our previous studies have shown that the
flanked Lmx1b allele was deleted in Wnt1-Cre; Lmx1bflox/2 and Pet1-
Cre; Lmx1bflox/2 mice [5,14]. Although we failed to provide direct
morphological data showing the deletion of Lmx1b, based on the
data mentioned above and phenotypes observed in Lmx1b iCKO
mice, it is reasonable to speculate that Lmx1b is inactivated in 5-
HTergic neurons of Lmx1b iCKO mice. Pet1-CreERT2 mice are
very useful in time-controlled deletion of interested genes in
central 5-HTergic neurons particularly in adulthood.
The function of Lmx1b in the development of 5-HTergic
neurons has been studied extensively [5,11,12,26]. In Lmx1b null
mice, postmitotic 5-HTergic neurons fail to express 5-HT and
several genes (e.g. Pet1) critical for 5-HT neuron development
[11,12]. When Lmx1b is conditionally deleted after 5-HTergic
neuron development has initiated (around embryonic day 12.5), 5-
HTergic neurons differentiate normally, but end up dying at later
embryonic stages [5,26]. Thus, Lmx1b is required for both
differentiation and survival of 5-HTergic neurons during embry-
onic development. As we showed in the present study, the
inactivation of Lmx1b in adulthood led to a reduction in central 5-
HT levels, probably as a consequence of Tph2 down-regulation. In
addition, Sert, the protein responsible for the re-uptake of 5-HT
into axonal terminals, and Vmat2, which is involved in packaging
5-HT into synaptic vesicles [22,23], were both greatly reduced in
the raphe nuclei of Lmx1b iCKO mice. In contrast, the expression
of both Pet1 and Aadc appeared unchanged in Lmx1b iCKO mice
relative to control mice, indicating that there was no loss of 5-
HTergic neurons in the raphe nuclei. It has been shown that Pet1
is required for terminal differentiation of 5-HTergic neurons
during embryonic development [4], and its expression is lost in
Lmx1b null mice [12]. Recently, it is reported that Pet1 is required
for maintaining the serotonergic neurotransmitter system during
adult stages [27]. Loss of Pet1 in the 5-HTergic neurons leads to a
decrease of Tph2 expression but no change in Lmx1b expression.
In the present study, normal Pet1 expression was found in Lmx1b
iCKO mice. It is likely that Lmx1b and Pet1 act in parallel to
regulate central 5-HTergic system, the expression of Pet1 in adult
brain is independent of Lmx1b, and Pet1 is not involved in altera-
tions of gene expression in Lmx1b iCKO mice. Taken together,
these results indicate that Lmx1b is required for 5-HT bio-
synthesis and expression of several key genes associated with func-
tions of 5-HTergic neurons, but not their survival in adult brain.
Central 5-HT deficiency has been associated with some mental
disorders, such as depression and posttraumatic stress disorder
[28,29,30]. We previously generated Lmx1b iCKO mice in which
Lmx1b is deleted specifically in 5-HTergic neurons at embryonic
stage with the help of Pet1-Cre, and found that 5-HT level in brain
is less than 10% of that in wild-type mice. Interestingly, these mice
showed enhanced contextual fear memory [5]. On the other hand,
5-HT plays important roles in the development of nervous system
at embryonic stages and during early postnatal development, such
as axonal growth [31], spine formation [32] and barrel formation
in the somatosensory cortex [33]. It is likely that abrogating 5-HT
biosynthesis or 5-HT neuronal development with traditional
genetic ablation techniques might have uncontrolled pleiotropic
effects by interfering with the development of other brain systems.
The use of Lmx1b iCKO mice circumvents these complications by
allowing the brain to develop normally through to adulthood, and
they serve as a new mouse model to study mental disorders
associated with central 5-HT deficiency.
Supporting Information
Figure S1 The expression of truncated Lmx1b in Lmx1biCKO mice is comparable to that of full-length Lmx1b inwild-type control. (A) The expression of full-length Lmx1b in
dorsal raphe of wild-type control. (B) The expression of truncated
Lmx1b in dorsal raphe of Lmx1b iCKO mice. Scale bar, 100 mm.
(DOC)
Figure S2 Nissl staining shows no difference in themorphological features of the dorsal raphe nucleusbetween wild-type and Lmx1b iCKO mice. Scale bar,
100 mm.
(DOC)
Author Contributions
Conceived and designed the experiments: NNS HL YQD. Performed the
experiments: NNS JBX YH JYC LZ. Analyzed the data: NNS JBX.
Contributed reagents/materials/analysis tools: LG KPL. Wrote the paper:
NNS YQD.
References
1. Lowry CA, Hale MW, Evans AK, Heerkens J, Staub DR, et al. (2008)
Serotonergic systems, anxiety, and affective disorder: focus on the dorsomedialpart of the dorsal raphe nucleus. Ann N Y Acad Sci 1148: 86–94.
2. Jacobs BL, Azmitia EC (1992) Structure and function of the brain serotonin
system. Physiol Rev 72: 165–229.
3. Cordes SP (2005) Molecular genetics of the early development of hindbrain
serotonergic neurons. Clin Genet 68: 487–494.
4. Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek EA, et al.
(2003) Pet-1 ETS gene plays a critical role in 5-HT neuron development and
is required for normal anxiety-like and aggressive behavior. Neuron 37:233–247.
5. Dai JX, Han HL, Tian M, Cao J, Xiu JB, et al. (2008) Enhanced contextual fear
memory in central serotonin-deficient mice. Proc Natl Acad Sci U S A 105:11981–11986.
6. Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A (1998) FGF and
Shh signals control dopaminergic and serotonergic cell fate in the anterior neural
plate. Cell 93: 755–766.
7. Pattyn A, Simplicio N, van Doorninck JH, Goridis C, Guillemot F, et al. (2004)
Ascl1/Mash1 is required for the development of central serotonergic neurons.Nat Neurosci 7: 589–595.
8. Craven SE, Lim KC, Ye W, Engel JD, de Sauvage F, et al. (2004) Gata2
specifies serotonergic neurons downstream of sonic hedgehog. Development
131: 1165–1173.
9. Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, et al. (1999)Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic
hedgehog signalling. Nature 398: 622–627.
10. van Doorninck JH, van Der Wees J, Karis A, Goedknegt E, Engel JD, et al.(1999) GATA-3 is involved in the development of serotonergic neurons in the
caudal raphe nuclei. J Neurosci 19: RC12.
11. Cheng L, Chen CL, Luo P, Tan M, Qiu M, et al. (2003) Lmx1b, Pet-1, andNkx2.2 coordinately specify serotonergic neurotransmitter phenotype. J Neurosci
23: 9961–9967.
12. Ding YQ, Marklund U, Yuan W, Yin J, Wegman L, et al. (2003) Lmx1b is
essential for the development of serotonergic neurons. Nat Neurosci 6: 933–938.
Adult Deletion of Lmx1b Leads to Central 5-HT Loss
PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e15998
13. Young P, Qiu L, Wang D, Zhao S, Gross J, et al. (2008) Single-neuron labeling
with inducible Cre-mediated knockout in transgenic mice. Nat Neurosci 11:
721–728.
14. Guo C, Qiu HY, Huang Y, Chen H, Yang RQ, et al. (2007) Lmx1b is essential
for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and
cerebellum development in mice. Development 134: 317–325.
15. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter
strain. Nat Genet 21: 70–71.
16. Scott MM, Wylie CJ, Lerch JK, Murphy R, Lobur K, et al. (2005) A genetic
approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl
Acad Sci U S A 102: 16472–16477.
17. Dai JX, Hu ZL, Shi M, Guo C, Ding YQ (2008) Postnatal ontogeny of the
transcription factor Lmx1b in the mouse central nervous system. J Comp Neurol
509: 341–355.
18. Gutknecht L, Waider J, Kraft S, Kriegebaum C, Holtmann B, et al. (2008)
Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2
knockout mice. J Neural Transm 115: 1127–1132.
19. Gutknecht L, Kriegebaum C, Waider J, Schmitt A, Lesch KP (2009) Spatio-
temporal expression of tryptophan hydroxylase isoforms in murine and human
brain: convergent data from Tph2 knockout mice. Eur Neuropsychopharmacol
19: 266–282.
20. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates. San
Diego: Academic Press, xxv, 1 v. (various pagings).
21. Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004)
Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305: 217.
22. Blakely RD, Berson HE, Fremeau RT, Jr., Caron MG, Peek MM, et al. (1991)
Cloning and expression of a functional serotonin transporter from rat brain.
Nature 354: 66–70.
23. Fon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, et al. (1997) Vesicular
transport regulates monoamine storage and release but is not essential for
amphetamine action. Neuron 19: 1271–1283.
24. Koed K, Linnet K (2000) Opposing changes in serotonin and norepinephrine
transporter mRNA levels after serotonin depletion. Eur Neuropsychopharmacol10: 501–509.
25. Armstrong DM, Ross CA, Pickel VM, Joh TH, Reis DJ (1982) Distribution of
dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the ratmedulla oblongata: demonstrated by the immunocytochemical localization of
catecholamine biosynthetic enzymes. J Comp Neurol 212: 173–187.26. Zhao ZQ, Scott M, Chiechio S, Wang JS, Renner KJ, et al. (2006) Lmx1b is
required for maintenance of central serotonergic neurons and mice lacking
central serotonergic system exhibit normal locomotor activity. J Neurosci 26:12781–12788.
27. Liu C, Maejima T, Wyler SC, Casadesus G, Herlitze S, et al. (2010) Pet-1 isrequired across different stages of life to regulate serotonergic function. Nat
Neurosci 13: 1190–1198.28. Middlemiss DN, Price GW, Watson JM (2002) Serotonergic targets in
depression. Curr Opin Pharmacol 2: 18–22.
29. Coppen A (1967) The biochemistry of affective disorders. Br J Psychiatry 113:1237–1264.
30. Naughton M, Mulrooney JB, Leonard BE (2000) A review of the role ofserotonin receptors in psychiatric disorders. Hum Psychopharmacol 15:
397–415.
31. Bonnin A, Torii M, Wang L, Rakic P, Levitt P (2007) Serotonin modulates theresponse of embryonic thalamocortical axons to netrin-1. Nat Neurosci 10:
588–597.32. Yan W, Wilson CC, Haring JH (1997) 5-HT1a receptors mediate the
neurotrophic effect of serotonin on developing dentate granule cells. BrainRes Dev Brain Res 98: 185–190.
33. Gaspar P, Cases O, Maroteaux L (2003) The developmental role of serotonin:
news from mouse molecular genetics. Nat Rev Neurosci 4: 1002–1012.34. Carlen M, Meletis K, Goritz C, Darsalia V, Evergren E, et al. (2009) Forebrain
ependymal cells are Notch-dependent and generate neuroblasts and astrocytesafter stroke. Nat Neurosci 12: 259–267.
Adult Deletion of Lmx1b Leads to Central 5-HT Loss
PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e15998
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